Anti-infective and osteogenic compositions and methods of use

ABSTRACT

Cationic steroidal antimicrobials (“CSAs” or “ceragenins”) and methods of making and using the same. Particularly advantageous uses include treating infections in patient and sub-patient populations, especially those having a bone disease, a broken bone, a bone infection, or a bone implant.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/953,176 filed Mar. 14, 2014, the disclosure of which is incorporatedherein in its entirety.

BACKGROUND

1. Field of the Invention

The present application relates to pharmaceutical chemistry,biochemistry, and medicine, more particularly, cationic steroidalantimicrobial (“CSA”) compounds (or “ceragenins”) and CSA compositions.

2. Description of the Related Art

Endogenous antimicrobial peptides, such as the human cathelicidin LL-37,play key roles in innate immunity. LL-37 is found in airway mucus and isbelieved to be important in controlling bacterial growth in the lung.Antimicrobial peptides are found in organisms ranging from mammals toamphibians to insects to plants. The ubiquity of antimicrobial peptideshas been used as evidence that these compounds do not readily engenderbacterial resistance. In addition, considering the varied sequences ofantimicrobial peptides among diverse organisms, it is apparent that theyhave evolved independently multiple times. Thus, antimicrobial peptidesappear to be one of “Nature's” primary means of controlling bacterialgrowth. However, clinical use of antimicrobial peptides presentssignificant issues, including the relatively high cost of producingpeptide-based therapeutics, the susceptibility of peptides to proteasesgenerated by the host and by bacterial pathogens, and deactivation ofantimicrobial peptides by proteins and DNA in lung mucosa.

An attractive means of harnessing the antibacterial activities ofantimicrobial peptides without the issues delineated above is to developnon-peptide mimics of antimicrobial peptides that display the samebroad-spectrum antibacterial activity utilizing the same mechanism ofaction. Non-peptide mimics would offer lower-cost synthesis andpotentially increased stability to proteolytic degradation. In addition,control of water solubility and charge density may be used to controlassociation with proteins and DNA in lung mucosa.

With over 1,600 examples of antimicrobial peptides known, it is possibleto categorize the structural features common to them. While the primarysequences of these peptides vary substantially, morphologies adopted bya vast majority are similar. Those that adopt alpha helix conformationsjuxtapose hydrophobic side chains on one face of the helix with cationic(positively charged) side chains on the opposite side. Similarmorphology is found in antimicrobial peptides that form beta sheetstructures: hydrophobic side chains on one face of the sheet andcationic side chains on the other.

SUMMARY OF THE INVENTION

Disclosed herein are methods of treating or preventing an infection in asubject in need thereof, comprising administering to the subject acompound of Formula (I), or pharmaceutically acceptable salt thereof:

wherein,

Z₁ and Z₂ are independently selected from the group consisting ofoptionally substituted alkyl; optionally substituted alkenyl; andoptionally substituted alkynyl; or H, with the proviso that Z₁ and Z₂are not both H;

R₂, R₃, and R₄, are independently selected from the group consisting ofoptionally substituted protected or unprotected amino-C_(a)-alkyl;optionally substituted protected or unprotectedamino-C_(a)-alkylcarbonyl; and optionally substituted protected orunprotected amino-C_(a)-alkylthiocarbonyl; and

a is 2-5. Preferably neither Z₁ nor Z₂ is H.

In some embodiments, the subject is a vertebrate, and in some cases is amammal. In some embodiments, the subject is a human. In someembodiments, the subject is in need of osteogenesis and the compound ofFormula (I) promotes osteogenesis. In some embodiments, the subject hasa bone disease, a broken bone, a bone infection, or a bone implant. Insome embodiments, the subject has a bone disease selected from the groupconsisting of bone resorption, osteoarthritis, osteoporosis,osteomalacia, osteitis fibrosa cystica, osteochondritis dissecans,osteomalacia, osteomyelitis, osteopenia, osteonecrosis, and porotichyperostosis. In some embodiments, the subject has a broken bone and thebroken bone results from a traumatic fracture, a critical sized bonedefect, distraction osteogenesis, spine fusion surgery, jointreplacement, an orthopaedic implant (bone implant), or a biopsy. In someembodiments, the administration of the compound of Formula (1) treats orprevents a bone disease, a broken bone, a bone infection, or a boneimplant.

In some embodiments, the infection is a bacterial infection. In someembodiments, the infection is a drug-resistant bacterial infection. Insome embodiments, the infection is a staphylococcus or pseudomonasbacterial infection, non-limiting examples of which includeMethicillin-resistant Staphylococcus aureus (MRSA) andTobramycin-resistant Pseudomonas aeruginosa (TRPA). In some embodiments,the infection is a gram negative bacterial infection. In someembodiments, the infection is a bone infection. In some embodiments, theinfection is an S. aureus or P. aeruginosa infection. In someembodiments, Z₁ and Z₂ are optionally substituted alkyl.

In some embodiments, Z₁ and Z₂ are optionally substituted alkenyl. Insome embodiments, Z₁ and Z₂ are optionally substituted alkynyl. In someembodiments, Z₁ and Z₂ are independently selected from the groupconsisting of optionally substituted C₁-C₂₀-alkyl; optionallysubstituted C₃-C₂₀-alkenyl; and optionally substituted C₃-C₂₀-alkynyl.In some embodiments, Z₁ and Z₂ are independently selected from the groupconsisting of optionally substituted C₃-C₁₀-alkyl ; optionallysubstituted C₃-C₁₀-alkenyl; and optionally substituted C₃-C₁₀-alkynyl.In some embodiments, Z₁ and Z₂ are independently selected from the groupconsisting of optionally substituted C₅-C₈-alkyl; optionally substitutedC₅-C₈-alkenyl; and optionally substituted C₅-C₈-alkynyl. In someembodiments, Z₁ and Z₂ are optionally substituted C₅-C₈-alkyl. In someembodiments, Z₁ and Z₂ are unsubstituted C₅-C₈-alkyl.

In some embodiments, R₂, R₃, and R₄, are amino-C_(a)-alkyl oramino-C_(a)-alkylcarbonyl. In some embodiments, R₂, R₃, and R₄, areamino-C_(a)-alkyl. In some embodiments, R₂, R₃, and R₄, areamino-C_(a)-alkylcarbonyl.

In some embodiments, a is 2. In some embodiments, a is 3. In someembodiments, a is 4. In some embodiments, a is 5.

In some embodiments, the protected amino-C_(a)-alkyl and the protectedamino-C_(a)-alkylcarbonyl are protected with a group independentlyselected from Fmoc, Boc, allyl, acetyl, benzyl, or CBz. In someembodiments, R₂, R₃, and R₄, are independently selected from the groupconsisting of carbamate protected or unprotected amino-C_(a)-alkyl andcarbamate protected or unprotected amino-C_(a)-alkylcarbonyl. In someembodiments, R₂, R₃, and R₄, are independently selected from the groupconsisting of Fmoc protected or unprotected amino-C_(a)-alkyl and Fmocprotected or unprotected amino-C_(a)-alkylcarbonyl. In some embodiments,R₂, R₃, and R₄, are independently selected from the group consisting ofBoc protected or unprotected amino-C_(a)-alkyl and Boc protected orunprotected amino-C_(a)-alkylcarbonyl.

In some embodiments the compound of Formula (I) is administered inconjuction with bone morphogenic protein (BMP).

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is not CSA-13.

In some embodiments, the optional substituent is fluorine. In someembodiments, the compound is not optionally substituted.

In some embodiments, the pharmaceutically acceptable salt is ahydrochloride salt.

Additional features and advantages will be set forth in part in thedescription that follows, and in part will be obvious from thedescription, or may be learned by practice of the embodiments disclosedherein. The objects and advantages of the embodiments disclosed hereinwill be realized and attained by means of the elements and combinationsparticularly pointed out in the appended claims. It is to be understoodthat both the foregoing brief summary and the following detaileddescription are exemplary and explanatory only and are not restrictiveof the embodiments disclosed herein or as claimed.

BRIEF DESCRIPTION OF THE DRAWINGS

To further clarify the above and other advantages and features of thepresent invention, a more particular description of the invention willbe rendered by references to specific embodiments thereof, which areillustrated in the appended drawings. It is appreciated that thesedrawings depict only typical embodiments of the invention and aretherefore not to be considered limiting of its scope. The invention willbe described and explained with additional specificity and detailthrough the use of the accompanying drawings in which:

FIG. 1 is a graph showing bone volume by microCT of ectopic bone nodulesfor various treatments;

FIG. 2 includes photographs of microscope slides showing none nodulehistology;

FIG. 3 includes representative X-ray images of fractured femora;

FIGS. 4A-4C are graphical representations of infection rate and pin sliprate; and

FIGS. 5A-5D are graphs showing MicroCT results assessing bone and tissuevolume parameters of the bone fracture callus.

DETAILED DESCRIPTION

The embodiments disclosed herein will now be described by reference tosome more detailed embodiments, with occasional reference to anyapplicable accompanying drawings. These embodiments may, however, beembodied in different forms and should not be construed as limited tothe embodiments set forth herein. Rather, these embodiments are providedso that this disclosure will be thorough and complete, and will fullyconvey the scope of the embodiments to those skilled in the art.

Definitions:

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which these embodiments belong. The terminology used in thedescription herein is for describing particular embodiments only and isnot intended to be limiting of the embodiments. As used in thespecification and the appended claims, the singular forms “a,” “an,” and“the” are intended to include the plural forms as well, unless thecontext clearly indicates otherwise. All publications, patentapplications, patents, and other references mentioned herein areincorporated by reference in their entirety.

Terms and phrases used in this application, and variations thereof,especially in the appended claims, unless otherwise expressly stated,should be construed as open ended as opposed to limiting. As examples ofthe foregoing, the term “including” should be read to mean “including,without limitation,” “including but not limited to,” and the like; theterm “comprising” as used herein is synonymous with “including,”“containing,” or “characterized by,” and is inclusive or open-ended anddoes not exclude additional, unrecited elements or method steps; theterm “having” should be interpreted as “having at least”; the term“includes” should be interpreted as “includes but is not limited to”;the term “example” is used to provide exemplary instances of the item indiscussion, not an exhaustive or limiting list thereof; and use of termslike “preferably,” “preferred,” “desired,” or “desirable,” and words ofsimilar meaning should not be understood as implying that certainfeatures are critical, essential, or even important to the structure orfunction of the invention, but instead as merely intended to highlightalternative or additional features that may or may not be utilized in aparticular embodiment. In addition, the term “comprising” is to beinterpreted synonymously with the phrases “having at least” or“including at least”. When used in the context of a process, the term“comprising” means that the process includes at least the recited steps,but may include additional steps. When used in the context of acompound, composition or device, the term “comprising” means that thecompound, composition or device includes at least the recited featuresor components, but may also include additional features or componentsLikewise, a group of items linked with the conjunction “and” should notbe read as requiring that each and every one of those items be presentin the grouping, but rather should be read as “and/or” unless expresslystated otherwise. Similarly, a group of items linked with theconjunction “or” should not be read as requiring mutual exclusivityamong that group, but rather should be read as “and/or” unless expresslystated otherwise.

It is understood that, in any compound described herein having one ormore chiral centers, if an absolute stereochemistry is not expresslyindicated, then each center may independently be of R-configuration orS-configuration or a mixture thereof. Thus, the compounds providedherein may be enantiomerically pure, enantiomerically enriched, racemicmixture, diastereomerically pure, diastereomerically enriched, or astereoisomeric mixture. In addition it is understood that, in anycompound described herein having one or more double bond(s) generatinggeometrical isomers that can be defined as E or Z, each double bond mayindependently be E or Z a mixture thereof.

Likewise, it is understood that, in any compound described, alltautomeric forms are also intended to be included.

It is to be understood that where compounds disclosed herein haveunfilled valencies, then the valencies are to be filled with hydrogensor isotopes thereof, e.g., hydrogen-1 (protium) and hydrogen-2(deuterium).

It is understood that the compounds described herein can be labeledisotopically. Substitution with isotopes such as deuterium may affordcertain therapeutic advantages resulting from greater metabolicstability, such as, for example, increased in vivo half-life or reduceddosage requirements. Each chemical element as represented in a compoundstructure may include any isotope of said element. For example, in acompound structure a hydrogen atom may be explicitly disclosed orunderstood to be present in the compound. At any position of thecompound that a hydrogen atom may be present, the hydrogen atom can beany isotope of hydrogen, including but not limited to hydrogen-1(protium) and hydrogen-2 (deuterium). Thus, reference herein to acompound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

Unless otherwise indicated, all numbers expressing quantities ofingredients, reaction conditions, and so forth used in the specificationand claims are to be understood as being modified in all instances bythe term “about.” Accordingly, unless indicated to the contrary, thenumerical parameters set forth in the specification and attached claimsare approximations that may vary depending upon the desired propertiessought to be obtained by the present embodiments. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical parameter shouldbe construed in light of the number of significant digits and ordinaryrounding approaches.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the embodiments are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Every numerical range given throughoutthis specification and claims will include every narrower numericalrange that falls within such broader numerical range, as if suchnarrower numerical ranges were all expressly written herein. Where arange of values is provided, it is understood that the upper and lowerlimit, and each intervening value between the upper and lower limit ofthe range is encompassed within the embodiments.

As used herein, the singular forms “a,” “and,” and “the” include pluralreferents unless the context clearly indicates otherwise. Thus, forexample, reference to “a compound” or “a CSA” includes a plurality ofcompounds/CSAs and reference to “an infection” can include reference toone or more infections, and so forth.

As used herein, any “R” group(s) such as, without limitation, R₁, R₂,R₃, and R₄ represent substituents that can be attached to the indicatedatom. Unless otherwise specified, an R group may be substituted orunsubstituted.

As used herein, any “Z” group(s) such as, without limitation, Z₁ and Z₂represent substituents that can be attached to the indicated atom.Unless otherwise specified, an R group may be substituted orunsubstituted.

A “ring” as used herein can be heterocyclic or carbocyclic. The term“saturated” used herein refers to a ring having each atom in the ringeither hydrogenated or substituted such that the valency of each atom isfilled. The term “unsaturated” used herein refers to a ring where thevalency of each atom of the ring may not be filled with hydrogen orother substituents. For example, adjacent carbon atoms in the fused ringcan be doubly bound to each other. Unsaturation can also includedeleting at least one of the following pairs and completing the valencyof the ring carbon atoms at these deleted positions with a double bond.

Whenever a group is described as being “optionally substituted” thatgroup may be unsubstituted, or substituted with one, two, three or moreof the indicated substituents, which may be the same or different, eachreplacing a hydrogen atom, unless otherwise indicated. If nosubstituents are indicated, it is meant that the indicated “substituted”group may be substituted with one or more group(s) individually andindependently selected from alkyl, alkenyl, alkynyl, cycloalkyl,cycloalkenyl, cycloalkynyl, acylalkyl, alkoxyalkyl, aminoalkyl, aminoacid, aryl, heteroaryl, heteroalicyclyl, aralkyl, heteroaralkyl,(heteroalicyclyl)alkyl, hydroxy, protected hydroxyl, alkoxy, aryloxy,acyl, mercapto, alkylthio, arylthio, cyano, halogen (e.g., F, Cl, Br,and I), thiocarbonyl, O-carbamyl, N-carbamyl, O-thiocarbamyl,N-thiocarbamyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido,C-carboxy, protected C-carboxy, O-carboxy, isocyanato, thiocyanato,isothiocyanato, nitro, oxo, silyl, sulfenyl, sulfinyl, sulfonyl,haloalkyl, haloalkoxy, trihalomethanesulfonyl,trihalomethanesulfonamido, an amino, a mono-substituted amino group anda di-substituted amino group, alkylaminoalkyl, dialkylaminoalkyl,di(alkyl)aminoalkyl, cyanato, alkylaminoalkylamino-alkylamino,arylaminoalkyl, aminoalkyloxy, aminoalkyloxyalkyl, aminoalkylcarboxy,aminoalkylaminocarbonyl, aminoalkylcarboxamido, guanidinoalkyloxy,hydroxyalkyl and protected derivatives thereof unless otherwiseindicated. The substituent may be attached to the group at more than oneattachment point. For example, an aryl group may be substituted with aheteroaryl group at two attachment points to form a fused multicyclicaromatic ring system. Biphenyl and naphthalene are two examples of anaryl group that is substituted with a second aryl group.

As used herein, “alkylthio” refers to the formula —SR wherein R is analkyl as is defined above, such as “C₁-C₉ alkylthio” and the like,including but not limited to methylmercapto, ethylmercapto,n-propylmercapto, 1-methylethylmercapto (isopropyl-mercapto),n-butylmercapto, iso-butylmercapto, sec-butylmercapto,tert-butylmercapto, and the like.

A “nitro” group refers to an “—NO₂” group.

A “cyanato” group refers to an “—OCN” group.

An “isocyanato” group refers to a “—NCO” group.

A “thiocyanato” group refers to a “—SCN” group.

An “isothiocyanato” group refers to an “—NCS” group.

A “sulfenyl” group refers to an “—S—R” group, which may be substitutedor unsubstituted. In some embodiments, R is selected from hydrogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂₋C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “sulfinyl” group refers to an “—S(═O)—R” group, which may besubstituted or unsubstituted. In some embodiments, R is selected fromhydrogen, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂₋C₆ alkynyl, C₃-C₇ carbocyclyl,C₆-C₁₀ aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl,as defined herein.

A “sulfonyl” group refers to an “—SO₂R” group, which may be substitutedor unsubstituted. In some embodiments, R is selected from hydrogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂₋C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “S-sulfonamido” group refers to a “—SO₂NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

An “N-sulfonamido” group refers to a “—N(R_(A))SO₂R_(B)” group in whichR_(A) and R_(b) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

An “O-carbamyl” group refers to a “—OC(═O)NR_(A)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

An “N-carbamyl” group refers to an “—N(R_(A))OC(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

An “O-thiocarbamyl” group refers to a “—OC(═S)NR_(A)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

An “N-thiocarbamyl” group refers to an “—N(R_(A))OC(═S)R_(B)” group inwhich R_(A) and R_(B) are each independently selected from hydrogen,C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀aryl, 5-10 membered heteroaryl, and 5-10 membered heterocyclyl, asdefined herein.

A “C-amido” group refers to a “—C(═O)NR_(A)R_(B)” group in which R_(A)and R_(B) are each independently selected from hydrogen, C₁-C₆ alkyl,C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl, 5-10membered heteroaryl, and 5-10 membered heterocyclyl, as defined herein.

An “N-amido” group refers to a “—N(R_(A))C(═O)R_(B)” group in whichR_(A) and R_(B) are each independently selected from hydrogen, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₇ carbocyclyl, C₆-C₁₀ aryl,5-10 membered heteroaryl, and 5-10 membered heterocyclyl, as definedherein.

As used herein, “C_(zz)” or “C_(zz) to C_(zzz)” or “C_(zz)-C_(zzz)” inwhich “zz” and “zzz” are integers refer to the number of carbon atoms inthe recited structure, such as an alkyl, alkenyl or alkynyl group, orthe number of carbon atoms in the ring of a cycloalkyl, cycloalkenyl,cycloalkynyl, aryl, heteroaryl or heteroalicyclyl group. That is, thealkyl, alkenyl, alkynyl, ring of the cycloalkyl, ring of thecycloalkenyl, ring of the cycloalkynyl, ring of the aryl, ring of theheteroaryl or ring of the heteroalicyclyl can contain from “zz” to“zzz”, inclusive, carbon atoms. Thus, for example, a “C₁ to C₄ alkyl”group refers to all alkyl groups having from 1 to 4 carbons, that is,CH₃—, CH₃CH₂—, CH₃CH₂CH₂—, (CH₃)₂CH—, CH₃CH₂CH₂CH₂—, CH₃CH₂CH(CH₃)— and(CH₃)₃C—. If no “zz” and “zzz” are designated with regard to an alkyl,alkenyl, alkynyl, cycloalkyl cycloalkenyl, cycloalkynyl, aryl,heteroaryl or heteroalicyclyl group, the broadest range described inthese definitions is to be assumed.

Unless otherwise specified, as used herein, “alkyl” refers to a straightor branched hydrocarbon chain that comprises a fully saturated (nodouble or triple bonds) hydrocarbon group. The alkyl group may have 1 to25 carbon atoms (whenever it appears herein, a numerical range such as“1 to 25” refers to each integer in the given range; e.g., “1 to 25carbon atoms” means that the alkyl group may consist of 1 carbon atom, 2carbon atoms, 3 carbon atoms, etc., up to and including 25 carbon atoms,although the present definition also covers the occurrence of the term“alkyl” where no numerical range is designated). The alkyl group mayalso be a medium size alkyl having 1 to 18 carbon atoms. The alkyl groupcould also be a lower alkyl having 1 to 6 carbon atoms. The alkyl groupof the compounds may be designated as “C₄” or “C₁-C₄ alkyl” or similardesignations. By way of example only, “C₁-C₄ alkyl” indicates that thereare one to four carbon atoms in the alkyl chain, i.e., the alkyl chainis selected from methyl, ethyl, propyl, iso-propyl, n-butyl, iso-butyl,sec-butyl, and t-butyl. Typical alkyl groups include, but are in no waylimited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiarybutyl, pentyl and hexyl. The alkyl group may be substituted orunsubstituted.

As used herein, “alkenyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more double bonds. Thealkenyl group may have 2 to 25 carbon atoms (whenever it appears herein,a numerical range such as “2 to 25” refers to each integer in the givenrange; e.g., “2 to 25 carbon atoms” means that the alkenyl group mayconsist of 2 carbon atom, 3 carbon atoms, 4 carbon atoms, etc., up toand including 25 carbon atoms, although the present definition alsocovers the occurrence of the term “alkenyl” where no numerical range isdesignated). The alkenyl group may also be a medium size alkenyl having2 to 15 carbon atoms. The alkenyl group could also be a lower alkenylhaving 1 to 6 carbon atoms. The alkenyl group of the compounds may bedesignated as “C₄” or “C₂-C₄ alkyl” or similar designations. An alkenylgroup may be unsubstituted or substituted.

As used herein, “alkynyl” refers to an alkyl group that contains in thestraight or branched hydrocarbon chain one or more triple bonds. Thealkynyl group may have 2 to 25 carbon atoms (whenever it appears herein,a numerical range such as “2 to 25” refers to each integer in the givenrange; e.g., “2 to 25 carbon atoms” means that the alkynyl group mayconsist of 2 carbon atom, 3 carbon atoms, 4 carbon atoms, etc., up toand including 25 carbon atoms, although the present definition alsocovers the occurrence of the term “alkynyl” where no numerical range isdesignated). The alkynyl group may also be a medium size alkynyl having2 to 15 carbon atoms. The alkynyl group could also be a lower alkynylhaving 2 to 6 carbon atoms. The alkynyl group of the compounds may bedesignated as “C₄” or “C₂-C₄ alkyl” or similar designations. An alkynylgroup may be unsubstituted or substituted.

As used herein, “aryl” or “aromatic” refers to a carbocyclic (allcarbon) monocyclic or multicyclic aromatic ring system (including fusedring systems where two carbocyclic rings share a chemical bond) that hasa fully delocalized pi-electron system throughout all the rings. Thenumber of carbon atoms in an aryl group can vary. For example, the arylgroup can be a C₆-C₁₄ aryl group, a C₆-C₁₀ aryl group, or a C₆ arylgroup (although the definition of C₆-C₁₀ aryl covers the occurrence of“aryl” when no numerical range is designated). Examples of aryl groupsinclude, but are not limited to, benzene, naphthalene and azulene. Anaryl group may be substituted or unsubstituted.

As used herein, “heteroaryl” refers to a monocyclic or multicyclicaromatic ring system (a ring system with fully delocalized pi-electronsystem) that contain(s) one or more heteroatoms, that is, an elementother than carbon, including but not limited to, nitrogen, oxygen andsulfur. The number of atoms in the ring(s) of a heteroaryl group canvary. For example, the heteroaryl group can contain 4 to 14 atoms in thering(s), 5 to 10 atoms in the ring(s) or 5 to 6 atoms in the ring(s).Furthermore, the term “heteroaryl” includes fused ring systems where tworings, such as at least one aryl ring and at least one heteroaryl ring,or at least two heteroaryl rings, share at least one chemical bond.Examples of heteroaryl rings include, but are not limited to, furan,furazan, thiophene, benzothiophene, phthalazine, pyrrole, oxazole,benzoxazole, 1,2,3-oxadiazole, 1,2,4-oxadiazole, thiazole,1,2,3-thiadiazole, 1,2,4-thiadiazole, benzothiazole, imidazole,benzimidazole, indole, indazole, pyrazole, benzopyrazole, isoxazole,benzoisoxazole, isothiazole, triazole, benzotriazole, thiadiazole,tetrazole, pyridine, pyridazine, pyrimidine, pyrazine, purine,pteridine, quinoline, isoquinoline, quinazoline, quinoxaline, cinnoline,and triazine. A heteroaryl group may be substituted or unsubstituted.

As used herein, “aralkyl” and “aryl(alkyl)” refer to an aryl groupconnected, as a substituent, via a lower alkylene group. The aralkylgroup may have 6 to 20 carbon atoms (whenever it appears herein, anumerical range such as “6 to 20” refers to each integer in the givenrange; e.g., “6 to 20 carbon atoms” means that the aralkyl group mayconsist of 6 carbon atom, 7 carbon atoms, 8 carbon atoms, etc., up toand including 20 carbon atoms, although the present definition alsocovers the occurrence of the term “aralkyl” where no numerical range isdesignated). The lower alkylene and aryl group of an aralkyl may besubstituted or unsubstituted. Examples include but are not limited tobenzyl, 2-phenylalkyl, 3-phenylalkyl, and naphthylalkyl.

An “O-carboxy” group refers to a “RC(═O)O—” group in which R can behydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl,heteroaryl, heteroalicyclyl, aralkyl, (heteroaryl)alkyl or(heteroalicyclyl)alkyl, as defined herein. An O-carboxy may besubstituted or unsubstituted.

A “thiocarbonyl” group refers to a “—C(═S)R” group in which R can be thesame as defined with respect to O-carboxy. A thiocarbonyl may besubstituted or unsubstituted.

A “trihalomethanesulfonamido” group refers to an “X₃CS(O)₂N(R_(A))—”group wherein each X is a halogen, and R_(A) hydrogen, alkyl, alkenyl,alkynyl, cycloalkyl, cycloalkenyl, cycloalkynyl, aryl, heteroaryl,heteroalicyclyl, aralkyl, (heteroaryl)alkyl or (heteroalicyclyl)alkyl.

The term “azido” as used herein refers to a —N₃ group.

A “mercapto” group refers to an “—SH” group.

A “carbonyl” group refers to a C═O group. Unless otherwise indicated,the carbonyl group may be substituted to form any of a ketone, amide,aldehyde, urea, and carbamate.

“Lower alkylene groups” refer to a C₁-C₂₅ straight-chained alkyltethering groups, such as —CH₂-tethering groups, forming bonds toconnect molecular fragments via their terminal carbon atoms. Examplesinclude but are not limited to methylene (—CH₂—), ethylene (—CH₂CH₂—),propylene (—CH₂CH₂CH₂—), and butylene (—CH₂CH₂CH₂CH₂—). A lower alkylenegroup can be substituted by replacing one or more hydrogen of the loweralkylene group with a substituent(s) listed under the definition of“substituted.” As used herein, “cycloalkyl” refers to a completelysaturated (no double or triple bonds) mono- or multi-cyclic hydrocarbonring system. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. Cycloalkyl groups can contain 3 to 10 atomsin the ring(s) or 3 to 8 atoms in the ring(s). A cycloalkyl group may beunsubstituted or substituted. Typical cycloalkyl groups include, but arein no way limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl and cyclooctyl.

As used herein, “cycloalkenyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more double bonds in atleast one ring; although, if there is more than one, the double bondscannot form a fully delocalized pi-electron system throughout all therings (otherwise the group would be “aryl,” as defined herein). Whencomposed of two or more rings, the rings may be connected together in afused fashion. A cycloalkenyl group may be unsubstituted or substituted.

As used herein, “cycloalkynyl” refers to a mono- or multi-cyclichydrocarbon ring system that contains one or more triple bonds in atleast one ring. If there is more than one triple bond, the triple bondscannot form a fully delocalized pi-electron system throughout all therings. When composed of two or more rings, the rings may be joinedtogether in a fused fashion. A cycloalkynyl group may be unsubstitutedor substituted.

As used herein, “alkoxy” or “alkyloxy” refers to the formula —OR whereinR is an alkyl, an alkenyl, an alkynyl, a cycloalkyl, a cycloalkenyl or acycloalkynyl as defined above. A non-limiting list of alkoxys aremethoxy, ethoxy, n-propoxy, 1-methylethoxy(isopropoxy), n-butoxy,iso-butoxy, sec-butoxy and tert-butoxy. An alkoxy may be substituted orunsubstituted.

As used herein, “acyl” refers to a hydrogen, alkyl, alkenyl, alkynyl,aryl, or heteroaryl connected, as substituents, via a carbonyl group.Examples include formyl, acetyl, propanoyl, benzoyl, and acryl. An acylmay be substituted or unsubstituted.

As used herein, “alkoxyalkyl” or “alkyloxyalkyl” refers to an alkoxygroup connected, as a substituent, via a lower alkylene group. Examplesinclude alkyl-O-alkyl- and alkoxy-alkyl- with the terms alkyl and alkoxydefined herein.

As used herein, “hydroxyalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a hydroxy group. Exemplaryhydroxyalkyl groups include but are not limited to, 2-hydroxyethyl,3-hydroxypropyl, 2-hydroxypropyl, and 2,2-dihydroxyethyl. A hydroxyalkylmay be substituted or unsubstituted.

As used herein, “haloalkyl” refers to an alkyl group in which one ormore of the hydrogen atoms are replaced by a halogen (e.g.,mono-haloalkyl, di-haloalkyl and tri-haloalkyl). Such groups include butare not limited to, chloromethyl, fluoromethyl, difluoromethyl,trifluoromethyl and 1-chloro-2-fluoromethyl, 2-fluoroisobutyl. Ahaloalkyl may be substituted or unsubstituted.

The term “amino” as used herein refers to a —NH₂ group.

As used herein, the term “hydroxy” refers to a —OH group.

A “cyano” group refers to a “—CN” group.

The term “azido” as used herein refers to a —N₃ group.

As used herein, “aminoalkyl” refers to an amino group connected, as asubstituent, via an alkylene group. Examples include H₂N-alkyl- with theterm alkyl defined herein.

As used herein, “alkylcarboxyalkyl” refers to an alkyl group connected,as a substituent, to a carboxy group that is connected, as asubstituent, to an alkyl group. Examples include alkyl-C(═O)O-alkyl- andalkyl-O—C(═O)-alkyl- with the term alkyl as defined herein.

As used herein, “alkylaminoalkyl” refers to an alkyl group connected, asa substituent, to an amino group that is connected, as a substituent, toan alkyl group. Examples include alkyl-NH-alkyl-, with the term alkyl asdefined herein.

As used herein, “dialkylaminoalkyl” or “di(alkyl)aminoalkyl” refers totwo alkyl groups connected, each as a substituent, to an amino groupthat is connected, as a substituent, to an alkyl group. Examples include

with the term alkyl as defined herein.

As used herein, “alkylaminoalkylamino” refers to an alkyl groupconnected, as a substituent, to an amino group that is connected, as asubstituent, to an alkyl group that is connected, as a substituent, toan amino group. Examples include alkyl-NH-alkyl-NH—, with the term alkylas defined herein.

As used herein, “alkylaminoalkylaminoalkylamino” refers to an alkylgroup connected, as a substituent, to an amino group that is connected,as a substituent, to an alkyl group that is connected, as a substituent,to an amino group that is connected, as a substituent, to an alkylgroup. Examples include alkyl-NH-alkyl-NH-alkyl-, with the term alkyl asdefined herein.

As used herein, “arylaminoalkyl” refers to an aryl group connected, as asubstituent, to an amino group that is connected, as a substituent, toan alkyl group. Examples include aryl-NH-alkyl-, with the terms aryl andalkyl as defined herein.

As used herein, “aminoalkyloxy” refers to an amino group connected, as asubstituent, to an alkyloxy group. Examples include H₂N-alkyl-O— andH₂N-alkoxy- with the terms alkyl and alkoxy as defined herein.

As used herein, “aminoalkyloxyalkyl” refers to an amino group connected,as a substituent, to an alkyloxy group connected, as a substituent, toan alkyl group. Examples include H₂N-alkyl-O-alkyl- andH₂N-alkoxy-alkyl- with the terms alkyl and alkoxy as defined herein.

As used herein, “aminoalkylcarboxy” refers to an amino group connected,as a substituent, to an alkyl group connected, as a substituent, to acarboxy group. Examples include H₂N-alkyl-C(═O)O— and H₂N-alkyl-O—C(═O)—with the term alkyl as defined herein.

As used herein, “aminoalkylamino” refers to an amino group connected, asa substituent, to an alkyl group connected, as a substituent, to anamino group. Examples include H₂N-alkyl-NH— with the term alkyl asdefined herein.

As used herein, “aminoalkylcarboxamido” refers to an amino groupconnected, as a substituent, to an alkyl group connected, as asubstituent, to a carbonyl group connected, as a substituent to an aminogroup. Examples include H₂N-alkyl-C(═O)—NH— with the term alkyl asdefined herein.

As used herein, “guanidinoalkyloxy” refers to a guanidinyl groupconnected, as a substituent, to an alkyloxy group. Examples include

with the terms alkyl and alkoxy as defined herein.

As used herein, “guanidinoalkylcarboxy” refers to a guanidinyl groupconnected, as a substituent, to an alkyl group connected, as asubstituent, to a carboxy group. Examples include

with the term alkyl as defined herein.

As used herein, “quaternaryammoniumalkylcarboxy” refers to a quaternizedamino group connected, as a substituent, to an alkyl group connected, asa substituent, to a carboxy group. Examples include

with the term alkyl as defined herein.

The term “halogen atom” or “halogen” as used herein, means any one ofthe radio-stable atoms of column 7 of the Periodic Table of theElements, such as, fluorine, chlorine, bromine and iodine.

Where the numbers of substituents is not specified (e.g. haloalkyl),there may be one or more substituents present. For example “haloalkyl”may include one or more of the same or different halogens.

As used herein, the term “amino acid” refers to any amino acid (bothstandard and non-standard amino acids), including, but not limited to,α-amino acids, β-amino acids, γ-amino acids and δ-amino acids. Examplesof suitable amino acids include, but are not limited to, alanine,asparagine, aspartate, cysteine, glutamate, glutamine, glycine, proline,serine, tyrosine, arginine, histidine, isoleucine, leucine, lysine,methionine, phenylalanine, threonine, tryptophan and valine. Additionalexamples of suitable amino acids include, but are not limited to,ornithine, hypusine, 2-aminoisobutyric acid, dehydroalanine,gamma-aminobutyric acid, citrulline, beta-alanine, alpha-ethyl-glycine,alpha-propyl-glycine and norleucine.

A linking group is a divalent moiety used to link one steroid to anothersteroid. In some embodiments, the linking group is used to link a firstCSA compound with a second CSA compound (which may be the same ordifferent). An example of a linking group is (C₁-C₁₀) alkyloxy-(C₁-C₁₀)alkyl.

Commonly understand nomenclature and molecule representations aredescribed and utilized throughout the specification. For example, askilled artisan would readily appreciate that the following twostructural representations are equivalent (wherein A, B, C, and D areunspecified functional groups):

The terms “P.G.” or “PG” or “protecting group” or “protecting groups” asused herein refer to any atom or group of atoms that is added to amolecule in order to prevent existing groups in the molecule fromundergoing unwanted chemical reactions. Examples of protecting groupmoieties are described in T. W. Greene and P. G. M. Wuts, ProtectiveGroups in Organic Synthesis, 3. Ed. John Wiley & Sons, 1999, and in J.F. W. McOmie, Protective Groups in Organic Chemistry Plenum Press, 1973,both of which are hereby incorporated by reference for the limitedpurpose of disclosing suitable protecting groups. The protecting groupmoiety may be chosen in such a way, that they are stable to certainreaction conditions and readily removed at a convenient stage usingmethodology known from the art. A non-limiting list of protecting groupsinclude benzyl; substituted benzyl; alkylcarbonyls and alkoxycarbonyls(e.g., t-butoxycarbonyl (Boc), acetyl, or isobutyryl);arylalkylcarbonyls and arylalkoxycarbonyls (e.g., benzyloxycarbonyl orCBz, fluorenylmethylcarbonyl or Fmoc); substituted methyl ether (e.g.methoxymethyl ether or MOM); substituted ethyl ether; a substitutedbenzyl ether; tetrahydropyranyl ether; silyls (e.g., trimethylsilyl,triethylsilyl, triisopropylsilyl, t-butyldimethylsilyl,triisopropylsilyloxy-methyl, [2-(trimethylsilyl)ethoxy]methyl ort-butyldiphenylsilyl); esters (e.g. benzoate ester); carbonates (e.g.methoxymethylcarbonate); sulfonates (e.g. tosylate or mesylate); acyclicketal (e.g. dimethyl acetal); cyclic ketals (e.g., 1,3-dioxane,1,3-dioxolanes, and those described herein); acyclic acetal; cyclicacetal (e.g., those described herein); acyclic hemiacetal; cyclichemiacetal; cyclic dithioketals (e.g., 1,3-dithiane or 1,3-dithiolane);orthoesters (e.g., those described herein) and triarylmethyl groups(e.g., trityl; monomethoxytrityl (MMTr); 4,4′-dimethoxytrityl (DMTr);4,4′,4″-trimethoxytrityl (TMTr); and those described herein).Amino-protecting groups are known to those skilled in the art, andcarbamates such as Fmoc, Boc, and CBz, as well as allyl, and acyl areparticularly preferred protecting groups. In general, the species ofprotecting group is not critical unless explicitly defined, providedthat it is stable to the conditions of any subsequent reaction(s) onother positions of the compound and can be removed at the appropriatepoint without adversely affecting the remainder of the molecule. Inaddition, a protecting group may be substituted for another aftersubstantive synthetic transformations are complete. Clearly, where acompound differs from a compound disclosed herein only in that one ormore protecting groups of the disclosed compound has been substitutedwith a different protecting group, that compound is within thedisclosure.

Methods of Treatment, Uses, and Medicaments

Infection is a major complication that can affect orthopedic operationsand can have a devastating effect on morbidity. In particular, boneinfections can be challenging to treat once they are established.Disclosed herein are methods of treating or preventing an infection in asubject in need thereof by the administration of one or more or cationicsteroidal antimicrobial (CSA) compounds (or ceragenins), orpharmaceutically acceptable salts thereof. Disclosed herein are also CSAcompositions comprising at least one CSA compound, or pharmaceuticallyacceptable sat thereof, for use in the treatment prevention ofinfections in subjects having a bone disease, a bone implant, or abroken bone. Some embodiments include methods of treating or preventinginfection while concurrently promoting osteogenesis in a subject. Someembodiments include methods of treating or preventing infection whileconcurrently treating or preventing a bone disease, a broken bone, abone infection, or a bone implant with the administration of one or moreCSA compounds, or pharmaceutically acceptable salts thereof.

In some embodiments, the CSA compositions or methods further compriseadministering at least one growth factor. The growth factor administeredmay be a bone growth factor, which may enhance osteogenesis in thesubject. In some embodiments, the bone growth factor is recombinant bonemorphogenetic protein. In some embodiments, the recombinant bonemorphogenetic protein is recombinant human bone morphogenetic protein.In some embodiments, the bone morphogenetic protein is BMP-2. In otherembodiments the bone morphogenetic protein is BMP-2, BMP-3, BMP-4,BMP-5, BMP-6, BMP-7, and/or a combination of any of the aforementionedBMPs. In some embodiments, the bone growth factor is INFUSE® BMP-2and/or OP-1 BMP-7. The interaction between the bone growth factor andCSA compound may be a synergistic interaction resulting in osteogenesisat levels higher than expected from individual treatment using only aCSA compound or only a growth factor. In some exemplary embodiments, themethod further comprises administering an osteogenic nutrient,osteogenic supplement, or combinations thereof.

In some embodiments, one or more CSA compounds are administered to asubject having a broken bone. Examples of broken bones include fractures(including traumatic fractures, stress fractures, and fracturescharacterized by partial breakages such as greenstick fractures);critical sized bone defects; distraction osteogenesis; surgical bonealterations (including spine fusion surgery); and bone disruptionresulting from a joint replacement, an orthopaedic implant (boneimplant), or a biopsy. In some embodiments, subject may have anon-healing bone conditions, such as fractures or surgical removal orgrafting of bones.

The compositions disclosed herein can be administered to any bone,anywhere it is desirable to promote bone healing or bone synthesis;e.g., the compositions can be administered to fracture fixation (such asfracture healing) in broken bones, or treatment of osteoporotic bone(i.e., to promote osteogenesis and strengthen the bone). Thecompositions are also useful to strengthen or repair osteoporotic bone,where there is not a lot of nascent bone material to start with. In thisexemplary embodiment, the compositions may promote acceleratedosteogenesis within the osteoporotic bone, thereby strengthening thebone. This can be accomplished with little or no destruction of theosteoporotic bone; e.g., the compositions may be simply coated onto theosteoporotic bone, or alternatively, used to fill in defects. Inaddition, the compositions can be used to reconstruct a segmental defectin the case of missing bone, e.g., following tumor resection,polytrauma, or combinations thereof. In general, the compositions can beadministered anywhere it is desired to promote osteogenesis within oradjacent to bone as well as treat, prevent, or combat infection,particularly lowering the risk of developing a bone infection.Consequently, the CSA compositions disclosed herein are well-suited forcombination therapy (ameliorating infection and promoting osteogenesis)as well as promoting individual mono-therapies.

In some embodiments, one or more CSA compounds are administered withadditional compounds that provide therapeutic effects towards bonediseases or broken bones. In some embodiments, the CSA compound isadministered with one or more bisphosphonates. Examples ofbisphosphonates include Etidronate, Elodronate, Tiladronate,Pamidronate, Neridronate, Olpadronate, Alendronate, Ibandronate,Residronate, and/or Zoledronate. In some embodiments, the CSA compoundis administered with calcium and/or vitamin D. In some embodiments, theCSA compound is administered with compounds for the treatment ofosteoporosis. In some embodiments, the CSA compound is administered withTeriparatide, strontium ranelate, raloxifene, and/or Denosumab. In someembodiments, the administration of a single CSA compound, orpharmaceutically acceptable salt thereof, provides efficacy for treatingor preventing infections as well as efficacy for treating or preventinga bone disease, a broken bone, a bone infection, or a bone implant. Insome embodiments, the administration of two or more CSA compounds, orpharmaceutically acceptable salt thereof, provides efficacy for treatingor preventing infections as well as efficacy for treating or preventinga bone disease, a broken bone, a bone infection, or a bone implant. Suchadministration may also promote healing and/or osteogenesis. Whenmultiple CSA compounds are administered, one or all administeredcompounds may have anti-infective properties as well as therapeuticallydesirable properties for treating or preventing a bone disease, a brokenbone, a bone infection, or a bone implant.

In some embodiments, the subject is a mammal. Examples of mammalsinclude horses, cattle, pigs, and humans. In some embodiments, thesubject is a human. In some embodiments, the subject is in need ofosteogenesis and the compound of Formula (I) promotes osteogenesis. Insome embodiments, the subject has a bone disease, a broken bone, a boneinfection, or a bone implant. In some embodiments, the subject has abone disease selected from the group consisting of bone resorption,osteoarthritis, osteoporosis, osteomalacia, osteitis fibrosa cystica,osteochondritis dissecans, osteomalacia, osteomyelitis, osteopenia,osteonecrosis, and porotic hyperostosis. In some embodiments, thesubject has a broken bone and the broken bone results from a traumaticfracture; a critical sized bone defect; distraction osteogenesis; spinefusion surgery; joint replacement; an orthopaedic implant; or a biopsy.Orthopaedic implants are well known in the art and replace a missingjoint or bone or support a damaged bone. Common types of implantsinclude pins, rods, screws and plates.

In some embodiments, the infection is a bacterial infection. In someembodiments, the infection is a drug-resistant bacterial infection. Insome embodiments, the infection is MRSA or TRPA. In some embodiments,the infection is a staphylococcus or pseudomonas bacterial infection. Insome embodiments, the infection is a gram negative bacterial infection.In some embodiments, the infection is a bone infection. In someembodiments, the infection is an S. aureus or P. aeruginosa infection.

Some embodiments are directed to methods of treating or preventing aninfection in a subject in need thereof, comprising administering to thesubject a compound of Formula (I), or pharmaceutically acceptable saltthereof:

wherein,

Z₁ and Z₂ are independently selected from the group consisting ofoptionally substituted alkyl; optionally substituted alkenyl; andoptionally substituted alkynyl;

R₂, R₃, and R₄, are independently selected from the group consisting ofoptionally substituted protected or unprotected amino-C_(a)-alkyl;optionally substituted protected or unprotectedamino-C_(a)-alkylcarbonyl; and optionally substituted protected orunprotected amino-C_(a)-alkylthiocarbonyl; and

a is 2-5.

In some embodiments, Z₁ and Z₂ are optionally substituted alkyl. In someembodiments, Z₁ and Z₂ are unsubstituted alkyl. In some embodiments, Z₁and Z₂ are optionally substituted alkenyl. In some embodiments, Z₁ andZ₂ are unsubstituted alkenyl. In some embodiments, Z₁ and Z₂ areoptionally substituted alkynyl. In some embodiments, Z₁ and Z₂ areunsubstituted alkynyl.

In some embodiments, Z₁ and Z₂ are independently selected from the groupconsisting of C₁-C₂₀-alkyl; C₃-C₂₀-alkenyl; and C₃-C₂₀-alkynyl. In someembodiments, Z₁ and Z₂ are independently selected from the groupconsisting of C₃-C₁₀-alkyl; C₃-C₁₀-alkenyl; and C₃-C₁₀-alkynyl. In someembodiments, Z₁ and Z₂ are independently selected from the groupconsisting of C₅-C₈-alkyl; C₅-C₈-alkenyl; and C₅-C₈-alkynyl. In someembodiments, Z₁ and Z₂ are C₅-C₈-alkyl. In some embodiments, Z₁ and Z₂are unsubstituted C₅-C₈-alkyl.

In some embodiments, R₂, R₃, and R₄, are amino-C_(a)-alkyl oramino-C_(a)-alkylcarbonyl. In some embodiments, R₂, R₃, and R₄, areamino-C_(a)-alkyl. In some embodiments, R₂, R₃, and R₄, areamino-C_(a)-alkylcarbonyl. In some embodiments, a is 2. In someembodiments, a is 3. In some embodiments, a is 4. In some embodiments, ais 5.

In some embodiments, the protected amino-C_(a)-alkyl and the protectedamino-C_(a)-alkylcarbonyl are protected with a group independentlyselected from Fmoc, Boc, allyl, acetyl, benzyl, or CBz. In someembodiments, R₂, R₃, and R₄, are independently selected from the groupconsisting of carbamate protected or unprotected amino-C_(a)-alkyl andcarbamate protected or unprotected amino-C_(a)-alkylcarbonyl. In someembodiments, R₂, R₃, and R₄, are independently selected from the groupconsisting of Fmoc protected or unprotected amino-C_(a)-alkyl and Fmocprotected or unprotected amino-C_(a)-alkylcarbonyl. In some embodiments,R₂, R₃, and R₄, are independently selected from the group consisting ofBoc protected or unprotected amino-C_(a)-alkyl and Boc protected orunprotected amino-C_(a)-alkylcarbonyl.

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is:

In some embodiments, the compound of Formula (I) is:

In some embodiments, the optional substituent is fluorine. In someembodiments, the compound is not optionally substituted.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

Some embodiments comprise a compound of Formula (I), or pharmaceuticallyacceptable salt thereof, and a pharmaceutically acceptable excipient.Pharmaceutically acceptable salts and excipients are described in moredetail below.

Pharmaceutically Acceptable Salts

The compounds and compositions disclosed herein are optionally preparedas pharmaceutically acceptable salts. The term “pharmaceuticallyacceptable salt” as used herein is a broad term, and is to be given itsordinary and customary meaning to a skilled artisan (and is not to belimited to a special or customized meaning), and refers withoutlimitation to a salt of a compound that does not cause significantirritation to an organism to which it is administered and does notabrogate the biological activity and properties of the compound. In someembodiments, the salt is an acid addition salt of the compound.Pharmaceutical salts can be obtained by reacting a compound withinorganic acids such as hydrohalic acid (e.g., hydrochloric acid orhydrobromic acid), sulfuric acid, nitric acid, and phosphoric acid.Pharmaceutical salts can also be obtained by reacting a compound with anorganic acid such as aliphatic or aromatic carboxylic or sulfonic acids,for example formic acid, acetic acid, propionic acid, glycolic acid,pyruvic acid, malonic acid, maleic acid, fumaric acid, trifluoroaceticacid, benzoic acid, cinnamic acid, mandelic acid, succinic acid, lacticacid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinicacid, methanesulfonic acid, ethanesulfonic acid, p-toluensulfonic acid,salicylic acid, stearic acid, muconic acid, butyric acid, phenylaceticacid, phenylbutyric acid, valproic acid, 1,2-ethanedisulfonic acid,2-hydroxyethanesulfonic acid, benzenesulfonic acid,2-naphthalenesulfonic acid, or naphthalenesulfonic acid. Pharmaceuticalsalts can also be obtained by reacting a compound with a base to form asalt such as an ammonium salt, an alkali metal salt, such as a lithium,sodium or a potassium salt, an alkaline earth metal salt, such as acalcium, magnesium or aluminum salt, a salt of organic bases such asdicyclohexylamine, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine,C₁-C₇ alkylamine, cyclohexylamine, dicyclohexylamine, triethanolamine,ethylenediamine, ethanolamine, diethanolamine, triethanolamine,tromethamine, and salts with amino acids such as arginine and lysine; ora salt of an inorganic base, such as aluminum hydroxide, calciumhydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, orthe like.

In some embodiments, the pharmaceutically acceptable salt is ahydrochloride salt. In some embodiments, the pharmaceutically acceptablesalt is a mono-hydrochloride salt, a di-hydrochloride salt, atri-hydrochloride salt, or a tetra-hydrochloride salt. Some embodimentsare directed to a sulfuric acid addition salt or sulfonic acid additionsalt of a CSA compound. In some embodiments, the sulfonic acid additionsalt is a disulfonic acid addition salt. In some embodiments, thesulfonic acid addition salt is a 1,5-naphthalenedisulfonic acid additionsalt. In some embodiments, the acid addition salt is a mono-additionsalt. In other embodiments, the acid addition salt is a di-additionsalt. In other embodiments, the acid addition salt is a tetra-additionsalt.

In some embodiments, the pharmaceutically acceptable salt is a benzoicacid salt. In other embodiments, the acid addition salt is abenzenesulphonic acid salt. In other embodiments, the acid addition saltis a citric acid salt. In other embodiments, the acid addition salt is afumaric acid salt. In other embodiments, the acid addition salt is agalactaric acid (mucic acid) salt. In other embodiments, the acidaddition salt is al-hydroxy-2-naphthoic acid salt. In other embodiments,the acid addition salt is a pamoic acid salt. In other embodiments, theacid addition salt is a phosphoric acid salt. In other embodiments, theacid addition salt is a succinic acid salt. In other embodiments, theacid addition salt is a L-tartaric acid salt.

Pharmaceutical Compositions

While it is possible for the compounds described herein to beadministered alone, it may be preferable to formulate the compounds aspharmaceutical compositions (e.g., formulations). As such, in yetanother aspect, pharmaceutical compositions useful in the methods anduses of the disclosed embodiments are provided. A pharmaceuticalcomposition is any composition that may be administered in vitro or invivo or both to a subject in order to treat or ameliorate a condition.In a preferred embodiment, a pharmaceutical composition may beadministered in vivo. A subject may include one or more cells ortissues, or organisms. In some exemplary embodiments, the subject is ananimal. In some embodiments, the animal is a mammal. The mammal may be ahuman or primate in some embodiments. A mammal includes any mammal, suchas by way of non-limiting example, cattle, pigs, sheep, goats, horses,camels, buffalo, cats, dogs, rats, mice, and humans.

As used herein the terms “pharmaceutically acceptable” and“physiologically acceptable” mean a biologically compatible formulation,gaseous, liquid or solid, or mixture thereof, which is suitable for oneor more routes of administration, in vivo delivery, or contact. Aformulation is compatible in that it does not destroy activity of anactive ingredient therein (e.g., a CSA compound or ceragenin), or induceadverse side effects that far outweigh any prophylactic or therapeuticeffect or benefit.

In an embodiment, the pharmaceutical compositions may be formulated withpharmaceutically acceptable excipients such as carriers, solvents,stabilizers, adjuvants, diluents, etc., depending upon the particularmode of administration and dosage form. The pharmaceutical compositionsshould generally be formulated to achieve a physiologically compatiblepH, and may range from a pH of about 3 to a pH of about 11, preferablyabout pH 3 to about pH 7, depending on the formulation and route ofadministration. In alternative embodiments, it may be preferred that thepH is adjusted to a range from about pH 5.0 to about pH 8. Moreparticularly, the pharmaceutical compositions may comprise atherapeutically or prophylactically effective amount of at least onecompound as described herein, together with one or more pharmaceuticallyacceptable excipients. Optionally, the pharmaceutical compositions maycomprise a combination of the compounds described herein, or may includea second active ingredient useful in the treatment or prevention ofbacterial infection (e.g., anti-bacterial or anti-microbial agents).

Formulations, e.g., for parenteral or oral administration, are mosttypically solids, liquid solutions, emulsions or suspensions, whileinhalable formulations for pulmonary administration are generallyliquids or powders, with powder formulations being generally preferred.A preferred pharmaceutical composition may also be formulated as alyophilized solid that is reconstituted with a physiologicallycompatible solvent prior to administration. Alternative pharmaceuticalcompositions may be formulated as syrups, creams, ointments, tablets,and the like.

Compositions may contain one or more excipients. Pharmaceuticallyacceptable excipients are determined in part by the particularcomposition being administered, as well as by the particular method usedto administer the composition. Accordingly, there exists a wide varietyof suitable formulations of pharmaceutical compositions (see, e.g.,Remington's Pharmaceutical Sciences).

Suitable excipients may be carrier molecules that include large, slowlymetabolized macromolecules such as proteins, polysaccharides, polylacticacids, polyglycolic acids, polymeric amino acids, amino acid copolymers,and inactive virus particles. Other exemplary excipients includeantioxidants such as ascorbic acid; chelating agents such as EDTA;carbohydrates such as dextrin, hydroxyalkylcellulose,hydroxyalkylmethylcellulose, stearic acid; liquids such as oils, water,saline, glycerol and ethanol; wetting or emulsifying agents; pHbuffering substances; and the like. Liposomes are also included withinthe definition of pharmaceutically acceptable excipients.

The pharmaceutical compositions described herein may be formulated inany form suitable for the intended method of administration. Whenintended for oral use for example, tablets, troches, lozenges, aqueousor oil suspensions, non-aqueous solutions, dispersible powders orgranules (including micronized particles or nanoparticles), emulsions,hard or soft capsules, syrups or elixirs may be prepared. Compositionsintended for oral use may be prepared according to any method known tothe art for the manufacture of pharmaceutical compositions, and suchcompositions may contain one or more agents including sweetening agents,flavoring agents, coloring agents and preserving agents, in order toprovide a palatable preparation.

Pharmaceutically acceptable excipients particularly suitable for use inconjunction with tablets include, for example, inert diluents, such ascelluloses, calcium or sodium carbonate, lactose, calcium or sodiumphosphate; disintegrating agents, such as cross-linked povidone, maizestarch, or alginic acid; binding agents, such as povidone, starch,gelatin or acacia; and lubricating agents, such as magnesium stearate,stearic acid or talc.

Tablets may be uncoated or may be coated by known techniques includingmicroencapsulation to delay disintegration and adsorption in thegastrointestinal tract and thereby provide a sustained action over alonger period. For example, a time delay material such as glycerylmonostearate or glyceryl distearate alone or with a wax may be employed.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample celluloses, lactose, calcium phosphate or kaolin, or as softgelatin capsules wherein the active ingredient is mixed with non-aqueousor oil medium, such as glycerin, propylene glycol, polyethylene glycol,peanut oil, liquid paraffin or olive oil.

In another embodiment, pharmaceutical compositions may be formulated assuspensions comprising a compound of the embodiments in admixture withat least one pharmaceutically acceptable excipient suitable for themanufacture of a suspension.

In yet another embodiment, pharmaceutical compositions may be formulatedas dispersible powders and granules suitable for preparation of asuspension by the addition of suitable excipients.

Excipients suitable for use in connection with suspensions includesuspending agents, such as sodium carboxymethylcellulose,methylcellulose, hydroxypropyl methylcellulose, sodium alginate,polyvinylpyrrolidone, gum tragacanth, gum acacia, dispersing or wettingagents such as a naturally occurring phosphatide (e.g., lecithin), acondensation product of an alkylene oxide with a fatty acid (e.g.,polyoxyethylene stearate), a condensation product of ethylene oxide witha long chain aliphatic alcohol (e.g., heptadecaethyleneoxycethanol), acondensation product of ethylene oxide with a partial ester derived froma fatty acid and a hexitol anhydride (e.g., polyoxyethylene sorbitanmonooleate); polysaccharides and polysaccharide-like compounds (e.g.dextran sulfate); glycoaminoglycans and glycosaminoglycan-like compounds(e.g., hyaluronic acid); and thickening agents, such as carbomer,beeswax, hard paraffin or cetyl alcohol. The suspensions may alsocontain one or more preservatives such as acetic acid, methyl and/orn-propyl p-hydroxy-benzoate; one or more coloring agents; one or moreflavoring agents; and one or more sweetening agents such as sucrose orsaccharin.

The pharmaceutical compositions may also be in the form of oil-in wateremulsions. The oily phase may be a vegetable oil, such as olive oil orarachis oil, a mineral oil, such as liquid paraffin, or a mixture ofthese. Suitable emulsifying agents include naturally-occurring gums,such as gum acacia and gum tragacanth; naturally occurring phosphatides,such as soybean lecithin, esters or partial esters derived from fattyacids; hexitol anhydrides, such as sorbitan monooleate; and condensationproducts of these partial esters with ethylene oxide, such aspolyoxyethylene sorbitan monooleate. The emulsion may also containsweetening and flavoring agents. Syrups and elixirs may be formulatedwith sweetening agents, such as glycerol, sorbitol or sucrose. Suchformulations may also contain a demulcent, a preservative, a flavoringor a coloring agent.

Additionally, the pharmaceutical compositions may be in the form of asterile injectable preparation, such as a sterile injectable aqueousemulsion or oleaginous suspension. This emulsion or suspension may beformulated according to the known art using those suitable dispersing orwetting agents and suspending agents which have been mentioned above.The sterile injectable preparation may also be a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, such as a solution in 1,2-propane-diol.

The sterile injectable preparation may also be prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution, and isotonic sodium chloride solution. Inaddition, sterile fixed oils may be employed as a solvent or suspendingmedium. For this purpose any bland fixed oil may be employed includingsynthetic mono- or diglycerides. In addition, fatty acids such as oleicacid may be used in the preparation of injectables.

The pharmaceutical composition may also be in the form of a solution ofa salt form of the active ingredient in an appropriate aqueous vehicle,such as water or isotonic saline or dextrose solution. Also contemplatedare compounds which have been modified by substitutions or additions ofchemical or biochemical moieties which make them more suitable fordelivery (e.g., increase solubility, bioactivity, palatability, decreaseadverse reactions, etc.), for example by esterification, glycosylation,PEGylation, and complexation.

Many therapeutics have undesirably short half-lives and/or undesirabletoxicity. Thus, the concept of improving half-life or toxicity isapplicable to various treatments and fields. Pharmaceutical compositionscan be prepared, however, by complexing the therapeutic with abiochemical moiety to improve such undesirable properties. Proteins area particular biochemical moiety that may be complexed with a CSAcompound for administration in a wide variety of applications. In someembodiments, one or more CSAs are complexed with a protein. In someembodiments, one or more CSA compounds are complexed with a protein toincrease the CSA compound's half-life. In other embodiments, one or moreCSA compounds are complexed with a protein to decrease the CSAcompound's toxicity. Albumin is a particularly preferred protein forcomplexation with a CSA compound. In some embodiments, the albumin isfat-free albumin.

With respect to the CSA therapeutic, the biochemical moiety forcomplexation can be added to the pharmaceutical composition as 0.25,0.5, 0.75, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50, or 100 weightequivalents, or a range bounded by any two of the aforementionednumbers, or about any of the numbers. In some embodiments, the weightratio of albumin to CSA compound is about 18:1 or less, such as about9:1 or less. In some embodiments, the CSA compound is coated withalbumin.

Alternatively, or in addition, non-biochemical compounds can be added tothe pharmaceutical compositions to reduce the toxicity of thetherapeutic and/or improve the half-life. Suitable amounts and ratios ofan additive that can reduce toxicity can be determined via a cellularassay. With respect to the CSA therapeutic, toxicity reducing compoundscan be added to the pharmaceutical composition as 0.25, 0.5, 0.75, 1,1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 10, 20, 50, or 100 weight equivalents,or a range bounded by any two of the aforementioned numbers, or aboutany of the numbers. In some embodiments, the toxicity reducing compoundis a cocoamphodiacetate such as Miranol® (disodium cocoamphodiacetate).In other embodiments, the toxicity reducing compound is an amphotericsurfactant. In some embodiments, the toxicity reducing compound is asurfactant. In other embodiments, the molar ratio of cocoamphodiacetateto CSA compound is between about 8:1 and 1:1, preferably about 4:1. Insome embodiments, the toxicity reducing compound is allantoin.

In some embodiments, a CSA composition is prepared utilizing one or moresufactants. In specific embodiments, the CSA compound is complexed withone or more poloxamer surfactants. Poloxamer surfactants are nonionictriblock copolymers composed of a central hydrophobic chain ofpolyoxypropylene (poly(propylene oxide)) flanked by two hydrophilicchains of polyoxyethylene (poly(ethylene oxide)). In some embodiments,the poloxamer is a liquid, paste, or flake (solid). Examples of suitablepoloxamers include those by the trade names Synperonics, Pluronics, orKolliphor. In some embodiments, one or more of the poloxamer surfactantin the composition is a flake poloxamer. In some embodiments, the one ormore poloxamer surfactant in the composition has a molecular weight ofabout 3600 g/mol for the central hydrophobic chain of polyoxypropyleneand has about 70% polyoxyethylene content. In some embodiments, theratio of the one or more poloxamer to CSA compound is between about50:1; about 40:1; about 30:1; about 20:1; about 10:1; about 5:1; about1:1; about 1:10; about 1:20; about 1:30; about 1:40; or about 1:50. Inother embodiments, the ratio of the one or more poloxamer to CSAcompound is between 50:1; 40:1; 30:1; 20:1; 10:1; 5:1; 1:1; 1:10; 1:20;1:30; 1:40; or 1:50. In some embodiments, the ratio of the one or morepoloxamer to CSA compound is between about 50:1 to about 1:50. In otherembodiments, the ratio of the one or more poloxamer to CSA compound isbetween about 30:1 to about 3:1. In some embodiments, the poloxamer isPluronic F127.

The amount of poloxamer may be based upon a weight percentage of thecomposition. In some embodiments, the amount of poloxamer is about 10%,15%, 20%, 25%, 30%, 35%, 40%, about any of the aforementioned numbers,or a range bounded by any two of the aforementioned numbers or theformulation. In some embodiments, the one or more poloxamer is betweenabout 10% to about 40% by weight of a formulation administered to thepatient. In some embodiments, the one or more poloxamer is between about20% to about 30% by weight of the formulation. In some embodiments, theformulation contains less than about 50%, 40%, 30%, 20%, 10%, 5%, or 1%of CSA, or about any of the aforementioned numbers. In some embodiments,the formulation containes less than about 20% by weight of CSA compound.

The above described poloxamer formulations are particularly suited forthe methods of treatment, device coatings, preparation of unit dosageforms (e.g., solutions, mouthwashes, injectables), etc.

In one embodiment, the compounds described herein may be formulated fororal administration in a lipid-based formulation suitable for lowsolubility compounds. Lipid-based formulations can generally enhance theoral bioavailability of such compounds.

As such, a pharmaceutical composition comprIn some embodimentss atherapeutically or prophylactically effective amount of a compounddescribed herein, together with at least one pharmaceutically acceptableexcipient selected from the group consisting of—medium chain fatty acidsor propylene glycol esters thereof (e.g., propylene glycol esters ofedible fatty acids such as caprylic and capric fatty acids) andpharmaceutically acceptable surfactants such as polyoxyl 40 hydrogenatedcastor oil.

In an alternative preferred embodiment, cyclodextrins may be added asaqueous solubility enhancers. Preferred cyclodextrins includehydroxypropyl, hydroxyethyl, glucosyl, maltosyl and maltotriosylderivatives of α-, β-, and γ-cyclodextrin. A particularly preferredcyclodextrin solubility enhancer is hydroxypropyl-o-cyclodextrin (BPBC),which may be added to any of the above-described compositions to furtherimprove the aqueous solubility characteristics of the compounds of theembodiments. In one embodiment, the composition comprIn someembodimentss about 0.1% to about 20% hydroxypropyl-o-cyclodextrin, morepreferably about 1% to about 15% hydroxypropyl-o-cyclodextrin, and evenmore preferably from about 2.5% to about 10%hydroxypropyl-o-cyclodextrin. The amount of solubility enhancer employedwill depend on the amount of the compound of the embodiments in thecomposition.

In some exemplary embodiments, a CSA compound comprises a multimer(e.g., a dimer, trimer, tetramer, or higher order polymer). In someexemplary embodiments, the CSA compounds can be incorporated intopharmaceutical compositions or formulations. Such pharmaceuticalcompositions/formulations are useful for administration to a subject, invivo or ex vivo. Pharmaceutical compositions and formulations includecarriers or excipients for administration to a subject.

Such formulations include solvents (aqueous or non-aqueous), solutions(aqueous or non-aqueous), emulsions (e.g., oil-in-water orwater-in-oil), suspensions, syrups, elixirs, dispersion and suspensionmedia, coatings, isotonic and absorption promoting or delaying agents,compatible with pharmaceutical administration or in vivo contact ordelivery. Aqueous and non-aqueous solvents, solutions and suspensionsmay include suspending agents and thickening agents. Suchpharmaceutically acceptable carriers include tablets (coated oruncoated), capsules (hard or soft), microbeads, powder, granules andcrystals. Supplementary active compounds (e.g., preservatives,antibacterial, antiviral and antifungal agents) can also be incorporatedinto the compositions.

Cosolvents and adjuvants may be added to the formulation. Non-limitingexamples of cosolvents contain hydroxyl groups or other polar groups,for example, alcohols, such as isopropyl alcohol; glycols, such aspropylene glycol, polyethyleneglycol, polypropylene glycol, glycolether; glycerol; polyoxyethylene alcohols and polyoxyethylene fatty acidesters. Adjuvants include, for example, surfactants such as, soyalecithin and oleic acid; sorbitan esters such as sorbitan trioleate; andpolyvinylpyrrolidone.

A pharmaceutical composition and/or formulation contains a total amountof the active ingredient(s) sufficient to achieve an intendedtherapeutic effect.

Dosages

The formulations may, for convenience, be prepared or provided as a unitdosage form. Preparation techniques include bringing into associationthe active ingredient (e.g., CSA/ceragenin) and a pharmaceuticalcarrier(s) or excipient(s). In general, formulations are prepared byuniformly and intimately associating the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product. For example, a tablet may be made bycompression or molding. Compressed tablets may be prepared bycompressing, in a suitable machine, an active ingredient (e.g., aCSA/ceragenin) in a free-flowing form such as a powder or granules,optionally mixed with a binder, lubricant, inert diluent, preservative,surface-active or dispersing agent. Molded tablets may be produced bymolding, in a suitable apparatus, a mixture of powdered compound (e.g.,CSA/ceragenin) moistened with an inert liquid diluent. The tablets mayoptionally be coated or scored and may be formulated so as to provide aslow or controlled release of the active ingredient therein.

Compounds (e.g., CSAs/ceragenins), including pharmaceutical formulationscan be packaged in unit dosage forms for ease of administration anduniformity of dosage. A “unit dosage form” as used herein refers to aphysically discrete unit suited as unitary dosages for the subject to betreated; each unit containing a predetermined quantity of compoundoptionally in association with a pharmaceutical carrier (excipient,diluent, vehicle or filling agent) which, when administered in one ormore doses, is calculated to produce a desired effect (e.g.,prophylactic or therapeutic effect or benefit). Unit dosage forms cancontain a daily dose or unit, daily sub-dose, or an appropriate fractionthereof, of an administered compound (e.g., CSA/ceragenin). Unit dosageforms also include, for example, capsules, troches, cachets, lozenges,tablets, ampules and vials, which may include a composition in afreeze-dried or lyophilized state; a sterile liquid carrier, forexample, can be added prior to administration or delivery in vivo. Unitdosage forms additionally include, for example, ampules and vials withliquid compositions disposed therein. Unit dosage forms further includecompounds for transdermal administration, such as “patches” that contactwith the epidermis of the subject for an extended or brief period oftime. The individual unit dosage forms can be included in multi-dosekits or containers. Pharmaceutical formulations can be packaged insingle or multiple unit dosage forms for ease of administration anduniformity of dosage.

Compounds (e.g., CSAs/ceragenins) can be administered in accordance withthe methods at any frequency as a single bolus or multiple dose e.g.,one, two, three, four, five, or more times hourly, daily, weekly,monthly, or annually or between about 1 to 10 days, weeks, months, orfor as long as appropriate. Exemplary frequencies are typically from 1-7times, 1-5 times, 1-3 times, 2-times or once, daily, weekly or monthly.Timing of contact, administration ex vivo or in vivo delivery can bedictated by the infection, pathogenesis, symptom, pathology or adverseside effect to be treated. For example, an amount can be administered tothe subject substantially contemporaneously with, or within about 1-60minutes or hours of the onset of a symptom or adverse side effect,pathogenesis, or vaccination. Long-acting pharmaceutical compositionsmay be administered twice a day, once a day, once every two days, threetimes a week, twice a week, every 3 to 4 days, or every week dependingon half-life and clearance rate of the particular formulation. Forexample, in an embodiment, a pharmaceutical composition contains anamount of a compound as described herein that is selected foradministration to a patient on a schedule selected from: twice a day,once a day, once every two days, three times a week, twice a week, andonce a week.

Localized delivery is also contemplated, including but not limited todelivery techniques in which the compound is implanted, injected,infused, or otherwIn some embodiments locally delivered. Localizeddelivery is characterized by higher concentrations of drug at the siteof desired action (e.g., the tumor or organ to be treated) versussystemic concentrations of the drug. Well-known localized delivery formscan be used, including long-acting injections; infusion directly intothe site of action; depot delivery forms; controlled or sustaineddelivery compositions; transdermal patches; infusion pumps; and thelike. The CSA compound can further be incorporated into a biodegradableor bioerodible material or be put into or on a medical device.

Doses may vary depending upon whether the treatment is therapeutic orprophylactic, the onset, progression, severity, frequency, duration,probability of or susceptibility of the symptom, the type pathogenesisto which treatment is directed, clinical endpoint desired, previous,simultaneous or subsequent treatments, general health, age, gender orrace of the subject, bioavailability, potential adverse systemic,regional or local side effects, the presence of other disorders or dinsome embodimentsases in the subject, and other factors that will beappreciated by the skilled artisan (e.g., medical or familial history).Dose amount, frequency or duration may be increased or reduced, asindicated by the clinical outcome desired, status of the infection,symptom or pathology, any adverse side effects of the treatment ortherapy. The skilled artisan will appreciate the factors that mayinfluence the dosage, frequency and timing required to provide an amountsufficient or effective for providing a prophylactic or therapeuticeffect or benefit. The exact dosage will be determined by thepractitioner, in light of factors related to the subject that requirestreatment. Dosage and administration are adjusted to provide sufficientlevels of the active agent(s) or to maintain the desired effect. It willbe appreciated that treatment as described herein includes preventing adisease, ameliorating symptoms, slowing disease progression, reversingdamage, or curing a disease.

The dosage may range broadly, depending upon the desired effects and thetherapeutic indication. Alternatively dosages may be based andcalculated upon the surface area of the patient, as understood by thoseof skill in the art. Although the exact dosage will be determined on adrug-by-drug basis, in most cases, some generalizations regarding thedosage can be made. The systemic daily dosage regimen for an adult humanpatient may be, for example, an oral dose of between 0.01 mg and 3000 mgof the active ingredient, preferably between 1 mg and 700 mg, e.g. 5 to200 mg. In some embodiments, the daily dosage regimen is 1 mg, 5 mg, 10,mg, 25 mg, 50 mg, 75 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg,400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, or about any ofthe aforementioned numbers or a range bounded by any two of theaforementioned numbers. The dosage may be a single one or a series oftwo or more given in the course of one or more days, as is needed by thesubject. In some embodiments, the compounds will be administered for aperiod of continuous therapy, for example for a week or more, or formonths or years. Doses tailored for particular types of cancers orparticular patients can be selected based, in part, on the GI₅₀, TGI,and LC₅₀ values set forth in the Examples that follow. Particularlypreferred formulations for oral dosage include tablet or solutions,particularly solutions compatible with IV administration or solutionscompatible with oral administration/use.

In instances where human dosages for compounds have been established forat least some condition, those same dosages may be used, or dosages thatare between about 0.1% and 500%, more preferably between about 25% and250% of the established human dosage. Where no human dosage isestablished, as will be the case for newly-discovered pharmaceuticalcompositions, a suitable human dosage can be inferred from ED₅₀ or ID₅₀values, or other appropriate values derived from in vitro or in vivostudies, as qualified by toxicity studies and efficacy studies inanimals.

In cases of administration of a pharmaceutically acceptable salt,dosages may be calculated as the free base. As will be understood bythose of skill in the art, in certain situations it may be necessary toadminister the compounds disclosed herein in amounts that exceed, oreven far exceed, the above-stated, preferred dosage range in order toeffectively and aggressively treat particularly aggressive diseases orconditions.

Dosage amount and interval may be adjusted individually to provideplasma levels of the active moiety which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). Forexample, therapeutic dosages may result in plasma levels of 0.05 μg/mL,0.1 μg/mL, 0.5 μg/mL, 1 μg/mL, 5 μg/mL, 10 μg/mL, 15 μg/mL, 20 μg/mL, 25μg/mL, 30 μg/mL, 35 μg/mL, 40 μg/mL, 45 μg/mL, 50 μg/mL, 55 μg/mL, 60μg/mL, 65 μg/mL, 70 μg/mL, 75 μg/mL, 80 μg/mL, 85 μg/mL, 90 μg/mL, 95μg/mL, 100 μg/mL, a range bounded by any two of the aforementionednumbers, or about any of the aforementioned numbers and ranges. In someembodiments, the therapeutic dose is sufficient to establish plasmalevels in the range of about 0.1 μg/mL to about 10 μg/mL. In otherembodiments, the therapeutic dose is sufficient to establish plasmalevels in the range of 1 μg/mL to 20 μg/mL. The MEC will vary for eachcompound but can be estimated from in vitro data. Dosages necessary toachieve the MEC will depend on individual characteristics and route ofadministration. However, HPLC assays or bioassays can be used todetermine plasma concentrations. Dosage intervals can also be determinedusing MEC value. Compositions should be administered using a regimenwhich maintains plasma levels above the MEC for 10-90% of the time,preferably between 30-90% and most preferably between 50-90%. In casesof local administration or selective uptake, the effective localconcentration of the drug may not be related to plasma concentration.

Compounds disclosed herein can be evaluated for efficacy and toxicityusing known methods. For example, the toxicology of a particularcompound, or of a subset of the compounds, sharing certain chemicalmoieties, may be established by determining in vitro toxicity towards acell line, such as a mammalian, and preferably human, cell line. Theresults of such studies are often predictive of toxicity in animals,such as mammals, or more specifically, humans. Alternatively, thetoxicity of particular compounds in an animal model, such as mice, rats,rabbits, or monkeys, may be determined using known methods. The efficacyof a particular compound may be established using several recognizedmethods, such as in vitro methods, animal models, or human clinicaltrials. When selecting a model to determine efficacy, the skilledartisan can be guided by the state of the art to choose an appropriatemodel, dose, route of administration and/or regime.

As described herein, the methods of the embodiments also include the useof a compound or compounds as described herein together with one or moreadditional therapeutic agents for the treatment of disease conditions.Thus, for example, the combination of active ingredients may be: (1)co-formulated and administered or delivered simultaneously in a combinedformulation; (2) delivered by alternation or in parallel as separateformulations; or (3) by any other combination therapy regimen known inthe art. When delivered in alternation therapy, the methods describedherein may comprise administering or delivering the active ingredientssequentially, e.g., in separate solution, emulsion, suspension, tablets,pills or capsules, or by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, e.g., serially, whereas insimultaneous therapy, effective dosages of two or more activeingredients are administered together. Various sequences of intermittentcombination therapy may also be used.

Co-Administration:

As used herein, “co-administration” means concurrently or administeringone substance followed by beginning the administration of a secondsubstance within 24 hours, 20 hours, 16 hours, 12 hours, 8 hours, 4hours, 1 hour, 30 minutes, 15 minutes, 5 minutes, 1 minute, a rangebounded by any two of the aforementioned numbers, and/or about any ofthe aforementioned numbers.

In some embodiments, one or more CSA/ceragenin are co-administered. Inother embodiments, the co-administration of CSA/ceragenin accounts fortheir therapeutic benefit. In some embodiments, co-administration isconcurrent.

Some embodiments are directed to the use of companion diagnostics toidentify an appropriate treatment for the patient. A companiondiagnostic is an in vitro diagnostic test or device that providesinformation that is essential for the safe and effective use of acorresponding therapeutic product. Such tests or devices can identifypatients likely to be at risk for adverse reactions as a result oftreatment with a particular therapeutic product. Such tests or devicescan also monitor responsiveness to treatment (or estimate responsivenessto possible treatments). Such monitoring may include schedule, dose,discontinuation, or combinations of therapeutic agents. In someembodiments, the CSA/ceragenin is selected by measuring a biomarker inthe patient. The term biomarker includes, but is not limited to, geneticregulation, protein levels, RNA levels, and cellular responses such ascytotoxicity.

Synthesis of CSA Compounds:

Compounds described herein can be prepared by known methods, especiallyknown methods previously published by the inventors. A skilled artisanwill readily understand that minor variations of starting materials andreagents may be utilized to prepare known and novel cationic steroidalantimicrobials. Schematically, for example, the preparation of certaincompounds can be accomplished according to Schemes 1 and 2 as follows:

Scheme 1: Ether-Containing CSA Compounds

Reagents: (a) LiA1H₄, THF, (b) PG, Et₃N, DMF, (c) allylbromide, NaH,THF, (d) O₃, CH₂Cl₂, MeOH; Me₂S, NaBH₄, (e) MsCl, CH₂Cl₂, Et₃N; NaN₃,DMSO, (f) Deprotection; MsCl, CH₂Cl₂, Et₃N; HNZ₁Z₂, (g) LiALH₄, THF.

As shown above, compound C-1 is converted to the tetra-ol, compound C-2,using a reducing reagent such as lithium aluminum hydride. Treatment ofC-2 with a protecting group (“PG”) reagent selectively protects theprimary alcohol providing compound C-3. A variety of alcohol protectinggroups may be used for this transformation. See, e.g., T. W. Greene andP. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley& Sons, 1999, and in J. F. W. McOmie, Protective Groups in OrganicChemistry Plenum Press, 1973. Subsequent treatment of C-3 withallylbromide provides the allyl-ether functionalized compound C-4.Treatment of C-4 with ozone and subsequent reduction provides tri-olC-5. C-5 is then treated with a reagent to create an oxygen-containingleaving group, such as methane sulfonyl chloride (“MsCl”), which issubsequently displaced upon treatment with nucleophilic azide resultingin the formation of C-6. The protection alcohol functionality of C-6 isdeprotected using standard conditions and treated with a leaving groupreagent as described above. The leaving group is then displaced bytreatment with an optionally substituted alkyl amine to provide C-7. Tofacilitate nucleophilic displacement of the meslate leaving group,HNZ₁Z₂ may optionally be converted to an anion prior to displacement.C-7 is then subjected to reduction, using standard conditions, to reducethe azido functional groups to amines, resulting in C-8. A skilledartisan will readily appreciate that this general synthetic scheme canbe modified to prepare the CSA compounds described herein, including CSAcompounds with substituents and functional groups that are differentfrom those generally described above. Furthermore, various nucleophilessuch as HNZ₁Z₂ are either commercially available or readily preparedusing known synthetic protocols from commercially available startingmaterials.

Scheme 2: Ester-Containing CSA Compounds

Reagents: (a) LiAlH₄, THF, (b) PG, Et₃N, DMF, (h) 3-PG-aminopropanoicacid, dicyclohexylcarbodiimide, N-hydroxysuccinimide, CH₂Cl₂, MeOH, (i)Deprotection; MsCl, CH₂Cl₂, Et₃N, (j) R—NH₂, CH₂Cl₂, Et₃N, (k)Deprotection.

As shown above, compound C-1 is converted to the tetra-ol, compound C-2,using a reducing reagent such as lithium aluminum hydride. Treatment ofC-2 with a protecting group (“PG”) reagent selectively protects theprimary alcohol providing compound C-3. A variety of alcohol protectinggroups may be used for this transformation. See, e.g., T. W. Greene andP. G. M. Wuts, Protective Groups in Organic Synthesis, 3. Ed. John Wiley& Sons, 1999, and in J. F. W. McOmie, Protective Groups in OrganicChemistry Plenum Press, 1973. Compound C-3 is subsequently treated withesterification reagents [such as dicyclohexylcarbodiimide andN-hydroxysuccinimide, followed by 3-PG-aminopropanoic acid (theprotected amine derivative of 3-aminopropanoic acid)] to providecompound C-9. Compound C-9 is deprotected using standard conditions andtreated with a reagent to form a suitable leaving group (e.g. mesylchloride) as described above, to provide C-10. Treatment of C-10 with anoptionally substituted alkyl amine provides compound C-11, which issubjected to standard amine-deprotection conditions to provide CSA C-12.To facilitate nucleophilic displacement of the meslate leaving group,HNZ₁Z₂ may optionally be converted to an anion prior to displacement. Askilled artisan will readily appreciate that this general syntheticscheme can be modified to prepare the CSAs described herein, includingCSAs with substituents and functional groups that are different fromthose generally described above. Furthermore, various nucleophiles suchas HNZ₁Z₂ are either commercially available or readily prepared usingknown synthetic protocols from commercially available startingmaterials.

Non-limiting examples of CSA compounds described in the presentapplication are generally illustrated in the table below. A skilledartisan will readily understand, in view of the present disclosure, thatthese CSAs can be prepared according to Schemes 1 or 2 above, or minorvariations that are within ordinary skill in the art.

CSA Structure

Using the above-described processes and procedures, additional CSAcompounds generally and specifically described herein are synthesizedand evaluated for their relative therapeutic efficacy. In someembodiments, the CSA compound is not CSA-13.

Compounds of the invention and precursors to the compounds according tothe invention are available commercially, e.g., from Sigma-Aldrich Co.,St. Louis; Mo.; and Research Plus, Inc., Manasquan, N.J. Other compoundsaccording to the invention can be synthesized according to methodsdisclosed herein and in the art.

In some embodiments defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or study of the present invention, suitable methods andmaterials are described herein.

All of the features disclosed herein may be combined in any combination.Each feature disclosed in the specification may be replaced by analternative feature serving a same, equivalent, or similar purpose. Insome embodiments, disclosed features (e.g., compound structures) are anexample of a genus of equivalent or similar features. All applications,publications, patents and other references, GenBank citations and ATCCcitations cited herein are incorporated by reference in their entirety.In case of conflict, the specification, including definitions, willcontrol.

As used herein, all numerical values or numerical ranges includeintegers within such ranges and fractions of the values or the integerswithin ranges unless the context clearly indicates otherwise. Thus, toillustrate, reference to a range of 90-100%, includes 91%, 92%, 93%,94%, 95%, 95%, 97%, etc., as well as 91.1%, 91.2%, 91.3%, 91.4%, 91.5%,etc., 92.1%, 92.2%, 92.3%, 92.4%, 92.5%, etc., and so forth. Referenceto a range of 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as wellas 1, 2, 3, 4, 5, 6, 7 minutes, etc., and so forth. Reference to a rangeof 0-72 hrs, includes 1, 2, 3, 4, 5, 6, 7 hrs, etc., as well as 1, 2, 3,4, 5, 6, 7 minutes, etc., and so forth. Reference to a range of doses,such as 0.1-1 μg/kg, 1-10 μg/kg, 10-25 μg/kg, 25-50 μg/kg, 50-100 μg/kg,100-500 μg/kg, 500-1,000 μg/kg, 1-5 mg/kg, 5-10 mg/kg, 10-20 mg/kg,20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg, includes0.11-0.9 μg/kg, 2-9 μg/kg, 11.5-24.5 μg/kg, 26-49 μg/kg, 55-90μg/kg,125-400 μg/kg, 750-800 μg/kg, 1.1-4.9 mg/kg, 6-9 mg/kg, 11.5-19.5mg/kg, 21-49 mg/kg, 55-90 mg/kg, 125-200 mg/kg, 275.5-450.1 mg/kg, etc.A series of ranges, for example, 1-10 μg/kg, 10-25 μg/kg, 25-50 μg/kg,50-100 μg/kg, 100-500 μg/kg, 500-1,000 μg/kg, 1-5 mg/kg, 5-10 mg/kg,10-20 mg/kg, 20-50 mg/kg, 50-100 mg/kg, 100-250 mg/kg, 250-500 mg/kg,includes 1-25 μg/kg, 10-25 μg/kg, 25-100 μg/kg, 100-1,000 μg/kg, 1-10mg/kg, 1-20 mg/kg etc.

A number of embodiments of the invention have been described.Nevertheless, one skilled in the art, without departing from the spiritand scope of the invention, can make various changes and modificationsof the invention to adapt it to various usages and conditions. Forexample, salts, esters, ethers and amides of invention compoundsdisclosed herein are within the scope of this invention. Accordingly,the following examples are intended to illustrate but not limit thescope of invention described in the claims.

EXAMPLES Anti-Infective Activities

Representative Procedure for Measuring MIC Values: A suspension isprepared of bacteria containing ˜10⁶CFU (colony forming units)/mL from aculture incubated in TSB at 37° C. for 24 hours. Aliquots of 1 mL of thesuspension are added to test tubes containing 1 mL TSB and incrementallyvaried concentrations of CSA compound. The samples are subjected tostationary incubation at 37° C. for 24 hours. Sample turbidity isdetermined by measuring absorption at 760 nm (HP 8453 UV-VisibleChemstation, Hewlett Packard). Additionally, an alliquot from each ofthe samples showing no measurable turbidity is subcultured on TSA plates(alliquots were diluted to provide fewer than 300 CFU). Colonies thatgrow on the subculture after overnight incubation are counted and thenumber of CFU/mL in the samples is calculated. The calculated values arecompared to the number of CFU/mL in the original inoculum. MIC valuesare determined as the concentrations of the studied compounds at whichthe number of CFU/mL remained constant or decreased after incubation for24 hours. CSA compounds were screened against methicillin-resistant S.aureus (MRSA) and tobramycin-resistant P. aeruginosa (TRPA).Representative data for CSA-90 and CSA-91 is provided in Table 1 below:

TABLE 1 MIC Values (μg/mL) Ceragenin S. aureus (MRSA) P. aeruginosa(TRPA) CSA-90 0.6 3.0 CSA-91 5.0 6.0Ceragenins such as CSA-90 and CSA-91 are also known to be effectiveanti-viral agents (See U.S. Pat. No. 7,754,705).

Mammalian Ectopic Bone Formation Model

Using a mouse model, 10 μg rhBMP-2 was delivered via a Medronicabsorbable collagen sponge (ACS). The effects of the addition of 25 μgand 250 μg ceragenin (CSA-90) were tested as was 250 μg ceragenin(CSA-90) dosed in the absence of rhBMP-2. Bone formation was examined byXR (Faxitron) and quantified by microCT (Skyscan 1174). Bone noduleswere also examined by histology. Scanning data confirmed increases inbone volume with the addition of the ceragenin (See FIG. 1).

As FIG. 1 illustrates for the ectopic bone formation model, rhBMP-2induced bone nodules as previously reported. Mean bone volume wassignificantly increased with the addition of both 25 μg CSA-90 (P=0.02)and 250 μg CSA-90 (P<0.01). The 250 μg CSA-90+rhBMP-2 treatment groupshowed a mean increase in bone volume of 3.2 fold by microCT overrhBMP-2 alone. Histological assessment showed no significant decrease inosteoclast number resulting from CSA-90 treatment, and the assessmentshowed a cortical shell and pseudo marrow space for rhBMP-2 treatedgroups and evidence of localized tissue mineralization with CSA-90treatment alone (FIG. 2). These data suggest that CSA-90 was promotingbone anabolism rather than suppressing bone resorption.

Mammalian Open Fracture Model:

A mammalian open fracture model was conducted using rats (n=42, n=41after anesthesia recovery) with and without bacteria inoculation, basedon the gap healing model of Chen et al. (J Bone Joint Surg Am.88-A:1510-23, 2006). In this model, infection was initiated by deliveryof 10⁴ CFU of pathogenic Staphlococcus aureus in ACS at the time ofsurgery. Pilot work (n=10 rats) found that a bacterial load of 10⁴ ledto non-union required euthanasia before 3 weeks. Animals receivedtreatment with 10 μg rhBMP-2 and/or 250 μg CSA-90 co-delivered via theACS implant. The group design is shown in Table 2.

TABLE 2 Group Procedure Treatment N 1 Open fracture None 6 2 Openfracture 10 μg rhBMP-2 6 3 Open fracture 250 μg CSA-90 6 4 Open fracture250 μg CSA-90 + 6 10 μg rhBMP-2 5 Open fracture 10⁴ cfu Staph 3 6 Openfracture 10⁴ cfu Staph + 10 μg 3 rhBMP-2 7 Open fracture 10⁴ cfu Staph +250 μg 6 CSA-90 8 Open fracture 10⁴ cfu Staph + 250 μg 6 CSA-90 + 10 μgrhBMP-2

The primary outcome measure was the number of animals culled withinfected fractures. Animal health and radiographic bone healing weremonitored by an experienced veterinarian in a blinded fashion toinoculation or treatment. Animals diagnosed as having progressivelydeteriorating condition with XRs showing evidence of infection wereculled and infection confirmed by culture swab post-mortem. Secondaryoutcome measures included union at 3 weeks and 6 weeks (excludingpin-slippage), and bone callus microCT measures at 6 weeks.

In the open fracture model, inoculation with S. aureus led to allanimals being culled with infections at ˜day 17 when given no treatmentor dosed with rhBMP-2 (n=3 per group). In the inoculated groups, withCSA-90 or CSA-90 +rhBMP-2 resulted in no infected culls (n=6 per group),with one animal from the CSA-90 group culled with a pin slip (noinfection seen in culture) (FIG. 3). At 3 weeks no unions were seen inthe control non-inoculated fractures. In contrast, the union rate was˜50% in the CSA-90 treated groups and 100% in the rhBMP-2 group (no S.aureus) and 100% in the CSA-90 +rhBMP-2 groups. By 6 weeks 50% of thecontrol open fractures and the union rate in the CSA-90 group had alsoincreased but not to 100% (FIGS. 4A-4C). MicroCT analysis showed an asignificant increase in tissue volume of the callus in the CSA-90+rhBMP-2 group compared to controls at 6 weeks, although callusremodeling was advanced in all groups by this time point (FIGS. 5A-5D).A table providing the infection and pin slips in culled animals isprovided in Table 3.

TABLE 3 Infected Cull Pin Slip # Infected # Pin Group Group ID Rate CullRate Culls Slips 1 Control 17% 17% 1/6 1/6 2 CSA 0% 0% 0/6 0/6 3 BMP 0%17% 0/6 1/6 4 CSA/BMP 0% 17% 0/6 1/6 5 Bacteria 100% 0% 3/3 0/3 6Bacteria/CSA 0% 20% 0/5 1/5 7 Bacteria/BMP 100% 0% 3/3 0/3 8Bacteria/BMP/ 0% 0% 0/6 0/6 CSA

Data Summation

These data show that CSA compounds dosed at the time of operation canhave a prophylactic effect and prevent the development of a persistentinfection that would otherwise lead to non-union. Moreover, the CSAcompounds represent a new class of antibiotics and their use would notcontribute to the resistance to other antibiotics. Few models oforthopedic infection exist in the literature and these studiesdemonstrate the disclosed model is reproducible and able to testantibiotic efficacy. These data also suggest that in addition to itsantibiotic activity, CSA compounds are able to promote bone healing. Inthe ectopic bone formation model the representative compound showedsynergy with rhBMP-2 to increase bone volume. In the fracture model therepresentative compound led to an increase in early union at 3 weeks.While rhBMP-2 led to greater and more rapid increases in union, rhBMP-2alone in the presence of bacteria did not promote union in this model.Synergy of rhBMP-2 was also seen with the representative compound interms of callus tissue volume. Indeed, the data indicate that therepresentative compound completely prevented facture repair failureresulting from bacterial inoculation. Furthermore, union data at 3 weeksshows evidence of improved union with the representative compound alone,with and without bacterial inoculation over non-inoculated and untreatedcontrols. These data support the concept of CSA compounds asdual-function orthopedic agent; the representative compound preventedinfection and promoted bone formation and repair, alone and incombination with rhBMP-2.

Although the foregoing has been described in some detail by way ofillustrations and examples for purposes of clarity and understanding, itwill be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present disclosure. Therefore, it should be clearly understood thatthe forms disclosed herein are illustrative only and are not intended tolimit the scope of the present disclosure, but rather to also cover allmodification and alternatives coming with the true scope and spirit ofthe invention.

1. A method of treating or preventing an infection in a subject in needthereof, comprising administering to the subject a CSA compound ofFormula (I), or pharmaceutically acceptable salt thereof:

wherein Z₁ and Z₂ are independently selected from the group consistingof optionally substituted alkyl; optionally substituted alkenyl; andoptionally substituted alkynyl, or H, with the proviso that Z₁ and Z₂are not both H; R₂, R₃, and R₄, are independently selected from thegroup consisting of optionally substituted protected or unprotectedamino-C_(a)-alkyl; optionally substituted protected or unprotectedamino-C_(a)-alkylcarbonyl; and optionally substituted protected orunprotected amino-C_(a)-alkylthiocarbonyl; and a is 2-5.
 2. The methodof claim 1, wherein the subject is a mammal.
 3. The method of claim 1,wherein the subject is a human.
 4. The method of claim 1, wherein thesubject is in need of osteogenesis and the CSA compound, orpharmaceutically acceptable salt thereof, promotes osteogenesis.
 5. Themethod of claim 1, wherein the subject has a bone disease, a brokenbone, a bone infection, or a bone implant.
 6. The method of claim 1,wherein the administration of the CSA compound, or pharmaceuticallyacceptable salt thereof, treats or prevents a bone disease, a brokenbone, a bone infection, or a bone implant.
 7. The method of claim 1,wherein the subject has a bone disease selected from the groupconsisting of bone resorption, osteoarthritis, osteoporosis,osteomalacia, osteitis fibrosa cystica, osteochondritis dissecans,osteomalacia, osteomyelitis, osteopenia, osteonecrosis, and porotichyperostosis.
 8. The method of claim 1, wherein the subject has a brokenbone and the broken bone results from a traumatic fracture; a criticalsized bone defect; distraction osteogenesis; spine fusion surgery; jointreplacement; an orthopaedic implant; or a biopsy.
 9. The method of claim1, wherein the infection is a bacterial infection.
 10. The method ofclaim 1, wherein the infection is a drug-resistant bacterial infection.11. The method of claim 1, wherein the infection isMethicillin-resistant Staphylococcus aureus (MRSA) orTobramycin-resistant Pseudomonas aeruginosa (TRPA).
 12. The method ofclaim 1, wherein the infection is a staphylococcus or pseudomonasbacterial infection.
 13. The method of claim 1, wherein the infection isa gram negative bacterial infection.
 14. The method of claim 1, whereinthe infection is a bone infection.
 15. The method of claim 1, whereinthe infection is an S. aureus or P. aeruginosa infection.
 16. The methodof claim 1, wherein Z₁ and Z₂ are optionally substituted alkyl.
 17. Themethod of claim 1, wherein Z₁ and Z₂ are optionally substituted alkenyl.18. The method of claim 1, wherein Z₁ and Z₂ are optionally substitutedalkynyl.
 19. The method of claim 1, wherein Z₁ and Z₂ are independentlyselected from the group consisting of optionally substitutedC₁-C₂₀-alkyl; optionally substituted C₃-C₂₀-alkenyl; and optionallysubstituted C₃-C₂₀-alkynyl.
 20. The method of claim 1, wherein Z₁ and Z₂are independently selected from the group consisting of optionallysubstituted C₃-C₁₀-alkyl; optionally substituted C₃-C₁₀-alkenyl; andoptionally substituted C₃-C₁₀-alkynyl.
 21. The method of claim 1,wherein Z₁ and Z₂ are independently selected from the group consistingof optionally substituted C₅-C₈-alkyl; optionally substitutedC₅-C₈-alkenyl; and optionally substituted C₅-C₈-alkynyl.
 22. The methodof claim 1, wherein Z₁ and Z₂ are optionally substituted C₅-C₈-alkyl.23. The method of claim 1, wherein Z₁ and Z₂ are unsubstitutedC₅-C₈-alkyl.
 24. The method of claim 1, wherein R₂, R₃, and R₄, areamino-C_(a)-alkyl or amino-C_(a)-alkylcarbonyl.
 25. The method of claim1, wherein R₂, R₃, and R₄, are amino-C_(a)-alkyl.
 26. The method ofclaim 1, wherein R₂, R₃, and R₄, are amino-C_(a)-alkylcarbonyl.
 27. Themethod of claim 24, wherein a is selected from 2, 3, 4, or
 5. 28. Themethod of claim 1, wherein the protected amino-C_(a)-alkyl and theprotected amino-C_(a)-alkylcarbonyl are protected with a groupindependently selected from Fmoc, Boc, allyl, acetyl, benzyl, or CBz.29. The method of claim 1, wherein R₂, R₃, and R₄, are independentlyselected from the group consisting of carbamate protected or unprotectedamino-C_(a)-alkyl and carbamate protected or unprotectedamino-C_(a)-alkylcarbonyl.
 30. The method of claim 1, wherein R₂, R₃,and R₄, are independently selected from the group consisting of Fmocprotected or unprotected amino-C_(a)-alkyl and Fmoc protected orunprotected amino-C_(a)-alkylcarbonyl.
 31. The method of claim 1,wherein R₂, R₃, and R₄, are independently selected from the groupconsisting of Boc protected or unprotected amino-C_(a)-alkyl and Bocprotected or unprotected amino-C_(a)-alkylcarbonyl.
 32. The method ofclaim 1, wherein the compound of Formula (I) is:


33. The method of claim 1, wherein the compound of Formula (I) is:


34. The method of claim 1, wherein the CSA compound is substituted withfluorine.
 35. The method of claim 1, wherein the compound is notsubstituted.
 36. The method of claim 1, wherein the pharmaceuticallyacceptable salt is a hydrochloride salt.
 37. The method of claim 1,wherein the compound of Formula (I) is


38. The method of claim 1, wherein the CSA compound, or pharmaceuticallyacceptable salt thereof, is administered together with an effectivesynergistic amount of a growth factor, preferably a bone morphogenicprotein.